The overall objective of this proposal is to define the mechanisms that control survival, proliferation and loss of interstitial cells of Cajal (ICC). Normal gastrointestinal motility requires intact networks of ICC. Loss of ICC is associated with several disorders of gastrointestinal motility. Despite the prominent role ICC play in the control of gastrointestinal motility, the mechanisms that regulate their survival and proliferation and loss are still largely unknown. Maintenance of ICC numbers requires a balance between survival and proliferation and loss. The overarching theme of our work is that 5HT, through specific 5HT receptors expressed on ICC, regulates the balance between survival and proliferation of ICC and loss of ICC. The PI will test this central hypothesis by the use of primary cultures and organotypic cultures of ICC, patch clamp techniques on freshly dissociated human ICC and cultured mouse ICC, muscle strips to simultaneously record mechanical activity and intracellular electrical activity from ICC, immunohistochemistry, Ca2+ imaging, laser capture microdissection, electroporation, Western blots, RT-PCR, single cell PCR, and quantitative PCR. To determine the role of 5HT in the regulation of survival, proliferation and loss of ICC three hypotheses will be tested: 1) 5HT increases the number of ICC; 2) 5HT1, 5HT2B, 5HT3 and 5HT7 receptors are expressed on ICC; and 3) 5HT regulates cell survival, proliferation and loss by activation of specific 5HT receptors expressed on ICC that modulate intracellular Ca2+ handling. These hypotheses are supported by preliminary data that show that 5HT markedly increases ICC number in cellular and organotypic cultures, that ICC proliferate, that ICC express death receptors and undergo apoptosis, a necessary physiological process to regulate tissue homeostasis, that specific 5HT receptors are expressed on human and mouse ICC and that 5HT regulates intracellular Ca2+ in ICC. Successful completion of the proposed studies has both basic significance and clinical impact. The results of the studies will lead to a better understanding of the basic mechanisms that regulate the number of ICC while at the same time provide a better understanding of the mechanisms that contribute to loss of ICC and the development of motility disorders associated with ICC loss. Indeed, based on our preliminary data, early, directed, 5HT-based treatment may be proposed as a mechanism to reverse loss of ICC in motility disorders.